Differing thermal sensitivities of physiological processes alter ATP allocation.

Changes in environmental temperature affect rate processes at all levels of biological organization. Yet the thermal sensitivity of specific physiological processes that affect allocation of the ATP pool within a species is less well understood. In this study of developmental stages of the Pacific oyster, Crassostrea gigas, thermal sensitivities were measured for growth, survivorship, protein synthesis, respiration and transport of amino acids and ions. At warmer temperatures, larvae grew faster but suffered increased mortality. An analysis of temperature sensitivity (Q10 values) revealed that protein synthesis, the major ATP-consuming process in larvae of C. gigas, is more sensitive to temperature change (Q10 value of 2.9±0.18) than metabolic rate (Q10 of 2.0±0.15). Ion transport by Na+/K+-ATPase measured in vivo has a Q10 value of 2.1±0.09. The corresponding value for glycine transport is 2.4±0.23. Differing thermal responses for protein synthesis and respiration result in a disproportional increase in the allocation of available ATP to protein synthesis with rising temperature. A bioenergetic model is presented illustrating how changes in growth and temperature affect allocation of the ATP pool. Over an environmentally relevant temperature range for this species, the proportion of the ATP pool allocated to protein synthesis increases from 35 to 65%. The greater energy demand to support protein synthesis with increasing temperature will compromise energy availability to support other essential physiological processes. Defining the trade-offs of ATP demand will provide insights into understanding the adaptive capacity of organisms to respond to various scenarios of environmental change.

Biochemical bases of growth variation during development: a study of protein turnover in pedigreed families of bivalve larvae (Crassostrea gigas).

Animal size is a highly variable trait regulated by complex interactions between biological and environmental processes. Despite the importance of understanding the mechanistic bases of growth, predicting size variation in early stages of development remains challenging. Pedigreed lines of the Pacific oyster (Crassostrea gigas) were crossed to produce contrasting growth phenotypes to analyze the metabolic bases of growth variation in larval stages. Under controlled environmental conditions, substantial growth variation of up to 430% in shell length occurred among 12 larval families. Protein was the major biochemical constituent in larvae, with an average protein-to-lipid content ratio of 2.8. On average, 86% of protein synthesized was turned over (i.e. only 14% retained as protein accreted), with a regulatory shift in depositional efficiency resulting in increased protein accretion during later larval growth. Variation in protein depositional efficiency among families did not explain the range in larval growth rates. Instead, changes in protein synthesis rates predicted 72% of growth variation. High rates of protein synthesis to support faster growth, in turn, necessitated greater allocation of the total ATP pool to protein synthesis. An ATP allocation model is presented for larvae of C. gigas that includes the major components (82%) of energy demand: protein synthesis (45%), ion pump activity (20%), shell formation (14%) and protein degradation (3%). The metabolic trade-offs between faster growth and the need for higher ATP allocation to protein synthesis could be a major determinant of fitness for larvae of different genotypes responding to the stress of environmental change.

Shifting Balance of Protein Synthesis and Degradation Sets a Threshold for Larval Growth Under Environmental Stress.

Exogenous environmental factors alter growth rates, yet information remains scant on the biochemical mechanisms and energy trade-offs that underlie variability in the growth of marine invertebrates. Here we study the biochemical bases for differential growth and energy utilization (as adenosine triphosphate [ATP] equivalents) during larval growth of the bivalve Crassostrea gigas exposed to increasing levels of experimental ocean acidification (control, middle, and high pCO2, corresponding to ∼400, ∼800, and ∼1100 µatm, respectively). Elevated pCO2 hindered larval ability to accrete both shell and whole-body protein content. This negative impact was not due to an inability to synthesize protein per se, because size-specific rates of protein synthesis were upregulated at both middle and high pCO2 treatments by as much as 45% relative to control pCO2. Rather, protein degradation rates increased with increasing pCO2. At control pCO2, 89% of cellular energy (ATP equivalents) utilization was accounted for by just 2 processes in larvae, with protein synthesis accounting for 66% and sodium-potassium transport accounting for 23%. The energetic demand necessitated by elevated protein synthesis rates could be accommodated either by reallocating available energy from within the existing ATP pool or by increasing the production of total ATP. The former strategy was observed at middle pCO2, while the latter strategy was observed at high pCO2. Increased pCO2 also altered sodium-potassium transport, but with minimal impact on rates of ATP utilization relative to the impact observed for protein synthesis. Quantifying the actual energy costs and trade-offs for maintaining physiological homeostasis in response to stress will help to reveal the mechanisms of resilience thresholds to environmental change.

Metabolic Cost of Protein Synthesis in Larvae of the Pacific Oyster (Crassostrea gigas) Is Fixed Across Genotype, Phenotype, and Environmental Temperature.

The energy made available through catabolism of specific biochemical reserves is constant using standard thermodynamic conversion equivalents (e.g., 24.0 J mg protein(-1)). In contrast, measurements reported for the energy cost of synthesis of specific biochemical constituents are highly variable. In this study, we measured the metabolic cost of protein synthesis and determined whether this cost was influenced by genotype, phenotype, or environment. We focused on larval stages of the Pacific oyster Crassostrea gigas, a species that offers several experimental advantages: availability of genetically pedigreed lines, manipulation of ploidy, and tractability of larval forms for in vivo studies of physiological processes. The cost of protein synthesis was measured in larvae of C. gigas for 1) multiple genotypes, 2) phenotypes with different growth rates, and 3) different environmental temperatures. For all treatments, the cost of protein synthesis was within a narrow range--near the theoretical minimum--with a fixed cost (mean ± one standard error, n = 21) of 2.1 ± 0.2 J (mg protein synthesized)(-1) We conclude that there is no genetic variation in the metabolic cost of protein synthesis, thereby simplifying bioenergetic models. Protein synthesis is a major component of larval metabolism in C. gigas, accounting for more than half the metabolic rate in diploid (59%) and triploid larvae (54%). These results provide measurements of metabolic cost of protein synthesis in larvae of C. gigas, an indicator species for impacts of ocean change, and provide a quantitative basis for evaluating the cost of resilience.

Experimental ocean acidification alters the allocation of metabolic energy.

Energy is required to maintain physiological homeostasis in response to environmental change. Although responses to environmental stressors frequently are assumed to involve high metabolic costs, the biochemical bases of actual energy demands are rarely quantified. We studied the impact of a near-future scenario of ocean acidification [800 µatm partial pressure of CO2 (pCO2)] during the development and growth of an important model organism in developmental and environmental biology, the sea urchin Strongylocentrotus purpuratus. Size, metabolic rate, biochemical content, and gene expression were not different in larvae growing under control and seawater acidification treatments. Measurements limited to those levels of biological analysis did not reveal the biochemical mechanisms of response to ocean acidification that occurred at the cellular level. In vivo rates of protein synthesis and ion transport increased ∼50% under acidification. Importantly, the in vivo physiological increases in ion transport were not predicted from total enzyme activity or gene expression. Under acidification, the increased rates of protein synthesis and ion transport that were sustained in growing larvae collectively accounted for the majority of available ATP (84%). In contrast, embryos and prefeeding and unfed larvae in control treatments allocated on average only 40% of ATP to these same two processes. Understanding the biochemical strategies for accommodating increases in metabolic energy demand and their biological limitations can serve as a quantitative basis for assessing sublethal effects of global change. Variation in the ability to allocate ATP differentially among essential functions may be a key basis of resilience to ocean acidification and other compounding environmental stressors.

Genetically Determined Variation in Developmental Physiology of Bivalve Larvae (Crassostrea gigas).

Understanding the complex interactions that regulate growth and form is a central question in developmental physiology. We used experimental crosses of pedigreed lines of the Pacific oyster, Crassostrea gigas, to investigate genetically determined variations in larval growth and nutrient transport. We show that (i) transport rates at 10 and 100 µM glycine scale differentially with size; (ii) size-specific maximum transport capacity (Jmax) is genetically determined; and (iii) Jmax serves as an early predictive index of subsequent growth rate. This relationship between genetically determined Jmax and growth suggests the potential use of transporter genes as biomarkers of growth potential. Analysis of the genome of C. gigas revealed 23 putative amino acid transporter genes. The complexity of gene families that underpin physiological traits has additional precedents in this species and others and warrants caution in the use of gene expression as a biomarker for physiological state. Direct in vivo measurements of physiological processes using species with defined genotypes are required to understand genetically determined variance of nutrient flux and other processes that regulate development and growth.

Separating the nature and nurture of the allocation of energy in response to global change.

Understanding and predicting biological stability and change in the face of rapid anthropogenic modifications of ecosystems and geosystems are grand challenges facing environmental and life scientists. Physiologically, organisms withstand environmental stress through changes in biochemical regulation that maintain homeostasis, which necessarily demands tradeoffs in the use of metabolic energy. Evolutionarily, in response to environmentally forced energetic tradeoffs, populations adapt based on standing genetic variation in the ability of individual organisms to reallocate metabolic energy. Combined study of physiology and genetics, separating "Nature and Nurture," is, thus, the key to understanding the potential for evolutionary adaptation to future global change. To understand biological responses to global change, we need experimentally tractable model species that have the well-developed physiological, genetic, and genomic resources necessary for partitioning variance in the allocation of metabolic energy into its causal components. Model species allow for discovery and for experimental manipulation of relevant phenotypic contrasts and enable a systems-biology approach that integrates multiple levels of analyses to map genotypic-to-phenotypic variation. Here, we illustrate how combined physiological and genetic studies that focus on energy metabolism in developmental stages of a model marine organism contribute to an understanding of the potential to adapt to environmental change. This integrative research program provides insights that can be readily incorporated into individual-based ecological models of population persistence under global change.

Developmental expression, differential hormonal regulation and evolution of thyroid and glucocorticoid receptor variants in a marine acanthomorph teleost (Sciaenops ocellatus).

Interactions between the thyroid hormone (TH) and corticosteroid (CS) hormone axes are suggested to regulate developmental processes in vertebrates with a larval phase. To investigate this hypothesis, we isolated three nuclear receptors from a larval acanthomorph teleost, the red drum (Sciaenops ocellatus), and established their orthologies as thraa, thrb-L and gra-L using phylogenomic and functional analyses. Functional characterization of the TH receptors in COS-1 cells revealed that Thraa and Thrb-L exhibit dose-dependent transactivation of a luciferase reporter in response to T3, while SoThraa is constitutively active at a low level in the absence of ligand. To test whether interactions between the TH and CS systems occur during development, we initially quantified the in vivo receptor transcript expression levels, and then examined their response to treatment with triiodothyronine (T3) or cortisol. We find that sothraa and sothrb-L are autoregulated in response to exogenous T3 only during early larval development. T3 did not affect sogra-L expression levels, nor did cortisol alter levels of sothraa or sothrb-L at any stage. While differential expression of the receptors in response to non-canonical ligand hormone was not observed under the conditions in this study, the correlation between sothraa and sogra-L transcript abundance during development suggests a coordinated function of the TH and CS systems. By comparing the findings in the present study to earlier investigations, we suggest that the up-regulation of thraa may be a specific feature of metamorphosis in acanthomorph teleosts.

The onset of cortisol synthesis and the stress response is independent of changes in CYP11B or CYP21 mRNA levels in larval red drum (Sciaenops ocellatus).

Although cortisol plays an important role in teleost development, the onset of cortisol production and the cortisol stress response in teleosts remain poorly understood. Here we have reported basal cortisol levels and the development of the cortisol stress response in larval red drum (Sciaenops ocellatus). We isolated partial nucleic acid sequences encoding two key corticosteroidogenic enzymes, CYP11B and CYP21 and assessed ontogenetic patterns of their mRNA levels relative to basal and stress-induced cortisol production. Basal cortisol was first detected 3 days post-hatch (DPH) and reached a maximum at 9 DPH. Cortisol did not increase in response to an acute stressor prior to 6 DPH. From 6 DPH forward, stress caused significant increases in larval cortisol content. Stress-induced cortisol levels in 6-9 DPH larvae were highest 1h post-stress. In larvae 11 DPH and older, the highest cortisol measurements occurred 0.5h post-stress. Elevated cortisol was still evident after 3h in 6 DPH larvae. From 11 DPH onward, basal cortisol levels were reestablished in larvae by 1h post-stress. CYP11B and CYP21 transcripts were detected in red drum 12h prior to hatching and in all post-hatch larvae examined. Changes in CYP11B and CYP21 mRNA levels did not occur in association with the ontogenetic appearance of cortisol, or the onset of the stress response. As larvae developed, the dynamics of the cortisol stress response matured from a low magnitude, slow recovery response, to a response similar to that observed in juvenile and adult fish.

Tissue- and sex-specific regulation of CYP19A1 expression in the Atlantic croaker (Micropogonias undulatus).

To better define the tissue- and sex-specific roles of aromatase in fishes, we have isolated a CYP19A1 cDNA sequence from a well-developed model of teleost reproduction, the Atlantic croaker (Micropogonias undulatus). This cDNA encodes a protein which has high identity (57-90%) to known CYP19A1 proteins and segregates with teleost CYP19A1 proteins in molecular phylogenetic analysis. In both sexes, the gene encoding Atlantic croaker CYP19A1 is expressed primarily in gonadal tissue, but also in the brain and other tissues at much lower levels, as determined relative to ribosomal 18S RNA expression by real-time quantitative RT-PCR. In females, the highest levels of CYP19A1 mRNA are found in the developing ovary compared to spawning, regressing and resting ovaries. In contrast, testicular CYP19A1 expression is lowest in developing testes and increases in spawning and regressing testes, although there were no statistically significant differences between stages. Brain CYP19A1 mRNA levels are lower in animals with developing gonads compared to spawning fish. In vitro treatment with human chorionic gonadotropin (10 IU/ml) for 6 or 24h increases CYP19A1 mRNA approximately 16- and 43-fold, respectively, in isolated Atlantic croaker ovarian follicles, but has no effect on CYP19A1 mRNA in testicular or brain minces. Six hour in vitro treatment with sex steroids (estradiol, testosterone or 17,20 beta,21-trihydroxy-4-pregnen-3-one; 290 nM) does not alter CYP19A1 mRNA in ovary, testis or brain. The regulation of CYP19A1 in the Atlantic croaker therefore differs in a tissue- and sex-specific manner.